ABSTRACT Members of the Toll-like receptor (TLR) family recognize various pathogen- and host tissue-derived molecules, and initiate immune responses physiologically via inflammatory processes. Exaggerated or prolonged TLR activation, however, leads to pathological inflammation as observed in a large number of etiologically diverse diseases, such as bacterial sepsis, autoimmune diseases and cancer. Despite the apparent need, no effective small molecule inhibitors of the canonical TLR signaling pathway are available, neither as probes to explore TLR biology nor as drugs to treat patients. The goal of this project is to identify small molecule inhibitors of TLR-specific signaling pathways. One factor impeding drug development is the signaling mechanism involved, i.e. a series of protein interactions that are inherently difficult for targeted drug development. Specifically, TLRs signal via the hierarchically acting proteins TIRAP, MyD88, IRAKs and TRAF6, the latter defining the border towards common, TLR-non-specific pathways. Thus, signaling events up- stream of TRAF6 represent the desirable ?drug target window?. We have developed a unique, cellular high throughput screening (HTS) platform that retains the advantages of phenotypic screening, i.e. the testing of complex responses in the natural environment of cells but, at the same time, mitigates the major disadvantage by eliminating non-specific compounds. Key feature of this system are drug-inducible (GyrB-fused) forms of TIRAP, MyD88 and TRAF6, whose responses reflect the ?signaling level? of compound activity. We have validated this approach using the St. Jude bioactive compound library, which allowed in consecutive screening steps facile elimination of non-specific hits (>99% of hits blocking TRAF6), and identification of one compound as selective TLR inhibitor. Using this compound as tool molecule, we established a hit advancement algorithm including SAR analysis, quantitative proteomics, protein interaction- and cellular thermal shift assays (CETSA), collectively validating this approach as discovery tool for TLR antagonists. Here we propose to perform a HTS of the St. Jude collection (~650,000 compounds) to identify a set of TLR inhibitory compounds with defined chemotype. The primary screen (single concentration) based on TIRAP-GyrB identifies compounds that act at any possible level of the TLR pathway. The secondary screen (dose-response) based on TIRAP-, MyD88- and TRAF6-GyrB cells defines the signaling level. Compounds inhibiting TRAF6 will be discarded as TLR-non- specific. Inhibition of TIRAP or MyD88 will define TLR-specific compounds. TLR-specific compounds will be validated by physiological TLR stimulation and prioritized based on chemoinformatics analysis and chemistry inspection. SAR analysis will be conducted to validate chemotype tractability based on available analogs and exploratory chemistry, and mentioned biochemical algorithm will be used to define mechanism of action and drug target. In summary, we expect to identify diverse chemotypes with defined, chemically tractable TLR-inhibitory activity as starting point for successful, future drug development.